Platelet Function Tests What When Why

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Platelet Function TestsWhat, When, How

Dr. R. ManchandaProf. & Director Pathology

KEM Hospital

Pune



PlateletDiscovery

• Existance of the tiny cell called platelets was under

dispute by light microscopy, which was resolved.

• German anatomist Max Schultze (1825-1874) who first

offered a description of the platelet as "spherules“.

• Giulio Bizzozero (1846-1901),

Named these «piastrine»,

i.e. small plates (later platelets)

and confirmed the role of

platelets in coagulation



Platelet

• Remarkable mammalian adaptation.

• Required for human survival to prevent and arrest bleeding.

• Ironically, in the past century, this haemostatic activity becamemaladaptive.

• Develop age-dependent progressive atherosclerosis.

• Major contribution to ischaemic thrombotic vascular disease,the leading cause of death worldwide.



Platelets

• Produced in the bone marrow from mature megakaryocytes.

• Circulate in blood as disc-shaped anucleate particles.

• 2–4 µm in diameter with a mean platelet volume of 7 to 9 fl.

• 150 – 450,000 / cmm

• The average lifespan of a platelet is about 5 to 9 days.

• Regulated by thrombopoietin, a hormone usually produced by the liver and

kidney.

• Each megakaryocyte produces between 5,000 and 10,000 platelets.

• Contain many storage granules.

• A continuous membrane structure. Diverse cell surface receptors

• Signaling molecules that direct platelet adhesion, activation, and

aggregation as well as coagulation

• Sequestered in the spleen & Liver



Platelets

Cytoplasmic granules:

1.Alpha granules

2.Dense bodies



Alpha granules

• Platelet factor 4 Heparinoid neutralisation

• Betathromboglobulin ? chemotaxis

• Thrombospondin? aggregation

• Platelet derived growth factor Mitogenesis, vessel repair

• vonwilliebrand factor Adhesion ,aggregation

• Fibrinogen Aggregation ,coagulation

• Factor V Prothrombinase activity

• Fibronectin Fibroblast and platelet adhesion

• Plasminogen activator inhibitor 1 Inhibition of fibrinolysis



Dense Bodies:

• ADP Aggregation ,vasoconstriction

• ATP Degrades to ADP

• 5 – HT Vasoconstriction , aggregation

• Calcium ?

• Pyrophosphate ?



Plasma Membrane:

Number of specific receptors, often glycoproteins (GPs) through

which platelets interact.

• Some important glycoproteins: GP1a Collagen adhesion

GP1b Subendothelial microfibril adhesion:

GP II b-IIIa Fibronogen binding, aggregation:

GP IV Thrombosponding binding GP V Thromin binding, aggregation

GP IX Platelet adhesion: part of GP – 1 b complex



Platelets

• Platelets play a key role in both hemostasis and thrombosis.

• Accurate measurement of platelet function is critical for identifying

patients withplatelet dysfunction

hyperfunction,

monitoring of modern antiplatelet therapy.

• majority of the 20th century the only means of assessing plateletfunction were a small number of fairly unreliable tests

- manual platelet count,

- inspection of the peripheral blood smear,- bleeding time.



Platelets

• A major problem

• difficulty in simulating hemostasis in vitro.

• platelets are sensitive to manipulation,

• prone to artifactual in vitro activation.

• Early attempts to simulate hemostasis in vitro included methods in

which platelets were counted before and after exposure to foreign

surfaces (eg, glass columns) or thrombus formation was monitored

within closed plastic tube loops.



Platelets Disorders

• Platelet disorders are the most common cause of

bleeding

• 1. Platelet number – Thrombocytopenia Thrombocytosis

• 2. Platelet Function defect



• Clinically suspected bleeding tendency.

• Following up an abnormal first line test.

• Acute haemostatic failure.



Begin with-

- Clinical history (congenital or acquired ? Sec to)

- Family history (Hemophilia, vWF)

- Family tree - Thorough clinical examination (Liver, spleen, LN )

- site ( Local or Haemostatic )

- Nature & frequency (spontaneous or

post traumatic, duration, amount,transfusion etc.) - Type ( petechia, purpura and mucosal

bleeding – platelet disorder.

hematoma, hemarthrosis-

coagulation disorder)



Defective Platelets Function

• A defect in function is suspected A defect in function is suspected

• if there is prolonged bleeding timeif there is prolonged bleeding time

• with or without skin or mucosal hemorrhagewith or without skin or mucosal hemorrhage

• in the presence of normal platelet count.in the presence of normal platelet count.

• acquired platelet dysfunctions can occur at any age

• range in severity from mild to life-threatening

haemorrhages



Disorders of Platelets Function

CongenitalCongenital

Rare

• Glanzman’s diseaseGlanzman’s disease

• Bernard Soluier’sBernard Soluier’s• Storage granules defectStorage granules defect

AcquiredAcquired

CommonCommon

• DrugsDrugs• UremiaUremia

• Myeloproliferative disordersMyeloproliferative disorders• Multiple myelomaMultiple myeloma



Screening Coagulogram

• Bleeding time ( Ivy’s method )

• Whole blood clotting time(Lee & White)

• Platelet count

• Platelet morphology

• Clot retraction

• Prothrombin Time ( P T )

• Activated partial thrombplastin time(APTT)

• Thrombin Time

• Clot solubility test




• Bleeding time …… ………… ( upto 7 min ) ( Ivy’s method )

• Clott ing time …… ………… ( 5 to 11 min )

(Lee & White)

• Platelet count …… ………… ( 150 – 450,000 /cmm )

• Platelet morphology ………. ( size & clumps )

• Clot retraction ………… ( good )

• Prothrombin Time – T …………. ( sec )

( P T ) - C …………. ( sec )

• Activated partial thrombplastin time - T … ( sec )

(aPTT) C… ( sec )

• Thrombin Time ………… T… ( sec )

C... ( sec )

• Clot solubility test …………… Insoluble

Impression -




• Bleeding time ( Ivy’s method )

• Whole blood clotting time(Lee & White)

• Platelet count

• Platelet morphology

• Clot retraction

• Prothrombin Time ( P T )

• Activated partial thrombplastin time(APTT)

• Thrombin Time

• Clot solubility test



• Bleeding time and clotting time extremely insensitive tests

- Bleeding time remains normal untilplatelet count drops to 30,000/cmm.

- Clotting time remains normal until factor

VIII or IX is less than 1 %.



Bleeding Time

• Template bleeding time

• Described by Duke in 1910 is the oldest test of platelet

function

• Surprisingly it remains in wide use in the UK

• There is considerable variation in methodology between

laboratories.



Bleeding time

• The BT is highly dependent on –

• operator technique,

• is subjective

•is influenced by patient variables unrelated to haemostasis, such asage, gender, haematocrit, vascular pattern, skin thickness and skin

temperature.

• The BT therefore has poor reproducibility, sensitivity and specificity,

as well as being invasive; for these reasons it is not recommended.



Laboratory Evaluation

• A reliably predictive “screening” test for platelet

dysfunction does not exist.

• Neither the bleeding time (BT) nor the platelet function

analyzer (PFA) is good for screening asymptomatic

persons



Bleeding

• Platelet count - 150 – 450,000 /cmm

• Thrombocytopenia - < 150,000 /cmm

- Spontaneous bleeding < 20,000 /cmm

( Giant platelet - ITP )

( Small - marrow failure synd.)

- will bleed on injury < 50,000 /cmm

- If h/o bleeding > 40,000 /cmm

( Suspect Platelet function defect )



Platelet disorders

are common bleeding disorders

Congenital - Number

Function

Acquired - Number

Function

• The diagnostic evaluation of platelet disorders challenges both

clinicians and clinical laboratories

• Testing for these conditions is

complex,

not well standardizedtime consuming.



Platelet count Risk of bleeding Examples

( x109/L)

<75 Primary hemostasis impaired After major surgery,

trauma

<50 Spontaneous bleeding mostly seen in skin

Petechiae, purpura

<20 Noticeable hemorrhage seen in skin and mucosa Epistaxis, gingival bleed

<10 Possible life-threatening mucosa and CNS Acute

hemorrhage GI hemorrhage,

intracranial hemorrhage



Laboratory tests

• Laboratory tests for platelet disorders may include:

• - Assessing platelet number and size [MPV]

- Assessing platelet morphology – blood film

- Screening tests of platelet function

Activated Clotting Time [ACT],

Bleeding Time [BT] and

PFA-100

- Light Transmission Aggregometry

- Assessment of platelet nucleotides

- Flow cytometry e.g. to quantitate the presence or

absence of platelet membrane glycoproteins

- More specialised investigations which are primarily

the province of research laboratories



LTA

• LTA is primarily regarded as the gold standard forplatelet function testing. Invented in the 1960s,

•LTA measures the formation of platelet aggregates

• As a suspension of platelets in plasma (platelet-richplasma [PRP]) is typically turbid, the formation of

aggregates due to activation by an agonist addedexogenously to the PRP results in increased lighttransmission through the platelet suspension.

• The extent of light transmission corresponds to theextent of platelet aggregation



• Venipuncture

• should only be collected from fasting and resting subjects

• refrained from smoking and caffeine ingestion on the dayof testing

• Avoid non-steroidal anti-inflammatory drugs for 7 to 10

days

• Herbal remedies, garlic, alcohol and certain foods may

also cause acquired platelet dysfunction

Investigation of a suspected disorder of



Investigation of a suspected disorder of

platelet function:

• Certain drugs and foods to be avoided for 7 days prior to test

• 12 hour fasting is advised

- Bleeding time

- Platelet count

- Platelet size distribution

- Examination of a stained blood film



Case

• 4 year old child

• Petechial rash on the body

• Bleeding from mouth• H/o fever 7 days ago

• No past history of significance

• No family history of bleeding tendency• On examination – no organomegaly





• Bleeding time …… ………… > 10 min ( upto 7 min )

( Ivy’s method )• Clotting time …… ………… 7 min 30 sec ( 5 to 11 min )

(Lee & White)• Platelet count …… ………… 10,000 ( 150 – 450,000 /cmm )• Platelet morphology ………. Giant platelets ( size & clumps )

• Clot retraction ………… Poor ( good )• Prothrombin Time – T …………. 13 ( sec )

( P T ) - C …………. 12 ( sec )• Activated partial thrombplastin t ime - T … 26 ( sec )

(aPTT) C… 24 ( sec )• Thrombin Time ………… T… 10 ( sec )

C... 10 ( sec )• Clot solubili ty test …………… Normal Insoluble

Impression - Thrombocytopenia. Other parameters within normal limits

Adv – Bone marrow examination

Bone marrow – Increased megakaryocytes, young forms

Impression : Peripheral platelet destruction ( ITP )



Giant Platelets

• Larger than normal

• Increase in the mean platelet volume (MPV)

• Up to 10% of normal platelets are 'giant'

• When more than 20% of platelets are giant, other conditions must be

considered



Giant Platelet

Causes

• Congenital

• Acquired



Acquired

Acquired - more common.

• Immune thrombocytopenic Purpura

• Thrombocytopenia due to sepsis & infections

• Myeloproliferative disorders

• lymphoproliferative disorders

• Massive hemorrhage

• Prosthetic heart valve

• Splenectomy

• Vasculitis

• SLE

• TTP

Inherited Giant Platelet Disorders



Inherited Giant Platelet Disorders• With structural defect

Glycoprotein abnormalities

Bernard-Soulier syndrome

Velocardiofacial syndrome

Association with mitral valve defect

Glycoprotein IV abnormalities

Calpain defect

Montreal platelet syndrome

Alpha granules Gray platelet syndrome

• With abnormal neutrophil inclusions

May-Hegglin anomaly

Sebastian syndrome

With systemic manifestations Hereditary macrothrombocytopenia with hearing loss

Epstein syndrome

Fechtner syndrome

• With no specific abnormalities

Mediterranean macrothrombocytopenia asymptomatic const itutional macrothrombocytopenia (ACMT) (Harris

Platelet Synd.)

C it l



Congenital

• Once considered rare.

• Now recognized with increasing frequency.

• Due to quantification of platelet number as a part of

routine blood testing.

• Have variable bleeding symptoms.

• A bleeding tendency disproportionate to the platelet

counts

• Often misdiagnosed as ITP

C it l



Congenital

• Clinical presentation is widely heterogeneous

• Ranging from an asymptomatic condition to a severe life-threatening bleeding tendency.

• Recognized within the first few weeks of life, to mild

conditions that may remain undetected even inadulthood.

• There are syndromic forms associated with physicalstigmata

Macrothrombocytopenia with leukocyte




inclusions/MYH9 disorders

• Rare disorders

• Triad of giant platelets,

• Thrombocytopenia,

• Dohle body-like cytoplasmic inclusions in granulocytes



Classification of neutrophil NMMHCA localization pattern in MYH9

disorders.

NMMHCA-positive granules

Number Shape Size (um)

Type I 1 or 2 oval spindle 0.5 2.0

Type II 3 20 circle oval <1

Type III 20 < circle <0.5




y p y


Phenotype Macrothrombocytopenia Leukocyte inclusions Alport syndrome*

May-Hegglin anomaly + + --

Sebastian syndrome + + --

Fechtner syndrome + + +

Epstein syndrome + -- +

* Nephritis, deafness and cataracts.

( Ref ; S. Kunishima, H. Saito Blood Reviews (2006) 20, 111–121 )




y p y


• Usually transmitted in an autosomal dominant manner.

• Approximately 20% of cases are considered to be sporadic.

• Although most patients with these disorders do not bleed, a

few bleed and need only occasional treatment.



Disorders of Platelet Function



• Hereditary:

• Plasma membrane defectsBernard-Soulier syndrome

Glanzmann’s thrombasthenia

Platelet- type von Williebrand’s disease

Defective response to collagen

Primary platelet coagulant defect

Disorders of Platelet Function

• Deficiency of storage organelles



• Dense body deficiency

Idiopathic (storage pool disease)

Hermansky – Pudlak syndrome

Wiskott- Aldrich syndrome Chediak - Higashi syndrome

Thrombocytopenia with absent radii



• Alpha granule deficiency Grey platelet syndrome

• Deficiency of dense bodies and alpha granules

• Defects of thrombaxane synthesis

Cyclo – oxygenase deficiency Thromboxane synthetase deficiency



• Defects of response to thrombaxane A2 and ionophores

• Miscellaneous

Montreal platelet syndrome Defects of response to adrenalin

Epstein’s syndrome

Acquired Disorders:



• Myeloproliferative disorders- Chronic myeloid leukaemia

- Polycythemia vera

- Myelofibrosis- Thrombocythaemia

• Acute leukaemias, preleukaemic states

• Renal disease ( Uremia )

• Dysproteinaemias

• Acquired storage pool deficiency



q g p y

- Disseminated intravascular coagulation

- Auto immune diseases

-Haemolytic – uraemic syndrome

- Thrombotic thrombytopenic purpura

- Renal transplant rejection

- Severe burns

- Valvular heart disease

- Cardiopulmonary bypass



• Chronic hypoglycaemia

- Glycogen storage disease type I

-Fructose 1,6- diphosphatase deficiency

• Bartter’s syndrome

• Drugs Acetylesalicylic acid



- Acetylesalicylic acid

- Indomethacin

- Sulphinpyrazone

- Phenylbutazone

- Dipyridamole

- Aminophylline

- Prostanoids

- Pennicillins

- Cephalosporins

- Heparin

- Dextrans

- Ethanol

- Radiographic contrast agents

- Clofibrate

- Phenothiazine

- Garlic

Case



• 65 yr.old male• Fever, wt. loss,

• Few blue patches patches on arm

• Bleeding gums , off & on

• Mild spleenomegaly

• CBC – Hb – 10.5 gm %

TLC - 9,800 / cmm

DLC - Occ myelo , meta, nRBC

Platelet – 76,000/cmm, MPV – 10 fl, few giant platelet

Bone marrow - MDS

Granulocytic series



Case



• A 7yr old male child.

• Referred for complaint of red to blue coloured patches on

lower limbs and arm since 4 months.

• These patches were spontaneous in onset, seen

intermittently and were self resolving.

Case



•There was no h/o of any previous episode of bleeding,history of umbilical cord bleeding was negative.

• Patient was not on aspirin and related drugs.

• SYSTEMIC EXAMINATION - NAD

• investigations- Hb-13.6 gm%

TLC-19,000 /cmm

Plt Count-2,64,000 DLC-P27/L28/E41/M04




• Bleeding time …… 15 ( upto 7 min )

( Ivy’s method )

• Clott ing time …… 10 ( 5 to 11 min )

(Lee & White)

• Platelet count …… 2,64,000 ( 150 – 450,000 /cmm )

• Platelet morphology Normal ( size & clumps )

• Clot retraction Good ( good )

• Prothrombin Time – T 15 ( sec )

( P T ) - C 13 ( sec )

• Activated partial thrombplastin time - T … 31 ( sec )

(aPTT) C… 30 ( sec )

• Thrombin Time ………… T… 10 ( sec )

C... 10 ( sec )

• Clot solubility test Normal Insoluble

Impression - To rule out Platelet function defect



• Platelet function tests revealed –

No aggregation with collagen.

Reduced aggregation with ADP.

Normal aggregation with ristocetin.

Diagnosis

Acquired Platelet Dysfunction with Eosinophilia.

( APDE )

Case



• 5 yr old girl

• Bleeding from gums

• Frequent nose bleed

• No significant family history

• Hb 9.0 gm %

• Platelet 2,80,000 /cmm



PBS & Bleeding




Bl di ti M th 20 ( t 7 i )



• Bleeding time …… More than 20 ( upto 7 min )

( Ivy’s method )• Clott ing time …… 7.30 ( 5 to 11 min )

(Lee & White)• Platelet count …… 2,80,000 ( 150 – 450,000 /cmm )• Platelet morphology no clumps ( size & clumps)

• Clot retraction poor ( good )• Prothrombin Time – T 12 ( sec )

( P T ) - C 12 ( sec )• Activated partial thrombplastin time - T …32 ( sec )

(aPTT) C… 31 ( sec )

• Thrombin Time ………… T… 11 ( sec ) C... 11 ( sec )• Clot solubi lity test ………Normal Insoluble

Impression - Adv Platelet function test

( No aggregation with ADP, collagen - Normal agg. With RistocetinHighly suggestive of Glanzmann Thrombasthenia

Adv. Mol studies

Global tests of platelet haemostatic function



• The most widely performed tests for screening plateletfunction disorders are currently

• - Template BT

• - Platelet Function Analyser (PFA-100 closure time )

• - Platelet aggregometer

• - Thromboelastography (TEG)

• - Rotational Thromboelastometry (ROTEM)

• ROTEM and TEG provide global tests of haemostasis

and platelet function and are mainly used within the

surgical setting.



• The utility of most of these assay systems including

TEG/ROTEM for the screening and diagnosis of platelet

function defects has not yet been examinedsystematically and their use for this application is

therefore not currently recommended.

Thrombelastography= TEG



• Analyzes the entire hemostasis process

• Non-invasive instrument designed to analyze a whole blood sample to

assess a patient’s clinical hemostatic condition

• Primarily used during surgical procedures

• Basic Principle

– Monitors hemostasis in its entirety

• Clot initiation through clot lysis

• Net effect of all hemostatic components interacting together

• Activated blood maximizes thrombin generation and platelet activation

in vitro

• Demonstrates hemostatic potential of a blood sample at a given point

The Utili ty of Platelet and Coagulation Testing of

Antithrombotics



• An increasing need for the standardization of platelet

function and coagulation testing for the assessment of

antithrombotic therapies

• Identify ideal laboratory testing and monitoring

procedures for acquired and inherited platelet function

defects

• Evaluating patient status when treated with existing or

emerging antithrombotics.



• Currently there are no instruments that reliably assess

the risk of bleeding.

• The challenges that routinely faced are the complexity of

physiology, the need for standardization of platelet

testing methodology

• The necessity for appropriate interpretation of the test

results.



Evaluating "new-onset" thrombocytopenia



Review bleeding history ITP Congenital

1. When was the onset of bleeding/bruising/petechiae? Recent Life-long

2. Have there been changes in general health? Evaluate changes No change

Any new medications?

3. Has there been "excessive" bleeding after minor trauma No Yes

during menses, surgeries, childbirth?

4. Are there any family members with increased bleeding No Yes

or thrombocytopenia?

5. Have there been previous normal platelet values? Yes No

6. What has been the response to treatment, Increased platelets Variable/small

(steroids IVIG, anti-D, splenectomy)? (approximately 80%) effect

7. Response to platelet transfusions Poor response/ Good increment/

short-lived normal survival

macrothrombocytopenia

peripheral blood smear



acquired causes ( ITP, MDS etc )

granulocyte inclusion bodies Dyserythropoiesis Grey Platelets Severe bleeding

+ -- GATA-1 sequencing GPS RIPA

NMMHCA IF GATA-1mutation Absent Increase

Abnormal Normal BSS vWD 2B

MYH9 disorders GPIb/IX FCM

No expression -50% exp

BSS BSS hetero

• Platelet count should be confirmed manually in a calculating chamber.



• Careful examination of a smear for morphological assessment of leukocytesand erythrocytes.

• If granulocyte inclusion bodies are obscure or absent, immunofluorescenceanalysis for neutrophil NMMHC -A ( nonmuscle myosin heavy chain-A )

localization is helpful to make a clear distinction.

• Flow cytometric analysis of platelet GP Ib/IX expression can differentiateBSS heterozygotes from patients with ‘‘true’’ isolatedmacrothrombocytopenia.

• Patients with congenital macrothrombocytopenia generally do not respondto standard ITP treatments,

Approach



• Family history and blood cell morphology analysis for discriminating AITP from inherited thrombocytopenia in children with isolated chronicthrombocytopenia.

• Bone marrow examination and search for specific autoantibodiesusing the MAIPA test are of little help.

• An isotopic platelet life span study, when available, should be

performed before considering splenectomy to exclude the diagnosis ofinherited thrombocytopenia, especially when steroids and/or IgG IVadministration failed to raise the platelet count.

• First acquired causes of macrothrombocytopenia, including ITP and

Approach



y g

myelodysplastic syndromes, should be ruled out.

• Complete history and physical examination should be carefully performed

• In syndromic forms, patients show complications of physical abnormalitiessuch as facial, cardiac, renal disease, deafness, cataracts skeletal anomalies

and/or mental retardation.

• If the patient previously had normal platelet counts, acquired rather

than congenital conditions are more likely to be the underlying cause.

• In inherited macrothrombocytopenias, platelet counts are constantlydecreased, ranging from as low as 10X109/l to near normal 150X109/l.

• On a peripheral blood smear, the majority of platelets are large, being similarto or larger than red blood cells or small lymphocytes

• In contrast, in patients with the much more common ITP, large platelets are present but the majority are of normal size

Approach



• It is important to make a proper diagnosis to avoid unnecessary treatment.

• Affected families should be educated about their diagnosis to avoid

unnecessary medications and potentially dangerous treatments forpresumed ITP

• When evaluating patients with refractory ITP or undifferentiated

thrombocytopenia, congenital macrothrombocytopenias should be includedin the differential diagnosis

Platelet Function Tests What When Why

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